Hi!

I am new to gene expression analysis and was looking for advice on analysing an experiment in which transcription might decrease genome-wide.

In this experiment, I have measured transcription by means of TT-seq and spiked in Drosophila cells to be able to detect the supposed decrease in transcription. The question I am trying to answer is whether there is a global decrease in transcription or not.

So far, I have analysed the data with DESeq2 using the least variable Drosophila genes (dm6) and applied it on my human counts (from featureCounts -t exons).

dds_dm6 <- DESeqDataSetFromMatrix(countData = counts_dm6,
                              colData = sample_info,
                              design = ~ treatment)
dds_dm6 <- estimateSizeFactors(dds_dm6)

dds_hg38 <- DESeqDataSetFromMatrix(countData = counts_hg38,
                              colData = sample_info,
                              design = ~ treatment)
sizeFactors(dds_hg38) <- sizeFactors(dds_dm6)

According to this analysis, the results suggest that ~900 genes are deregulated, which are less genes that what I would expect.

Am I applying the Spike-in normalisation correctly to see if there is a global change in transcription?

Thanks, Ana

If you have features you want to use for normalization, use

dds <- estimateSizeFactors(dds, controlGenes = ...)
# then DESeq()